Presenter: Özge Gizlenci
Affiliation: Babraham Institute

Alternative splicing (AS) plays a major role in the differentiation of immune cells during an immune response as 29% of AS genes are specific to the immune system. Although the role of AS is extensively investigated in T cells, its role in B cell activation is less characterised. We sought to develop a long-read technology, Oxford Nanopore Technologies (ONT), workflow to understand post-transcriptional regulation at both gene and isoform levels of human germinal centre B cells. As one of the challenges of ONT is the accurate computational analysis of isoforms, we developed the ‘Nexons’ pipeline to identify differentially spliced transcript variants using long-read sequencing. An in-depth analysis of splicing regulators with Nexons revealed that poison exons of splicing factors (e.g. SRSF3) were preferentially spliced out upon activation whereas naïve B cells expressed isoforms carrying poison exon, leading to nonsense-mediated mRNA decay. Moreover, we identified novel spliced variants of these genes, which were difficult to deconvolute using short-read data due to the limitations of short-read technology. Altogether, our findings validate the combination of Nexons with ONT cDNA-PCR sequencing as a suitable method for the identification and quantification of complex isoforms.